Release of the neurotransmitter dopamine mediates the reward for such adaptive activities as eating and sex. Drugs of abuse circumvent the normal stimulation of this pathway and induce dopamine release more directly. The development of drug tolerance, physical dependence and craving therefore provides a paradigm to examine the more general question of neural plasticity, its role in the reward pathway and its relationship to behavior. Amphetamines induce the efflux of dopamine into the synapse and appear to interact with the vesicular amine transporter. The behavioral effects of amphetamines further suggest that regulation of these transport activities may have a role in the normal function of reqard pathways and in behavior. Indeed, Dr. Sulzer has used voltammetry to show that stimulation of dopamine D2-like receptors reduces quantal size. Activity-dependent changes in neurotransmitter transport may also account for drug tolerance, dependence and craving. The long-term objectives of this program are to understand how the transport of neurotransmitters into synaptic vesicles influences dopamine release and the function of the reqard pathway. We originally used the neurotoxin MPP+_ to isolate the first cDNA for a vesicular neurotransmitter transporter, a transporter for monoamines. The sequences defines a novel mammalian gene family and now includes two vesicular amine and one vesicular acetylcholine transporter. The strategy of the proposal is to study the mechanism, regulation and cell biology of the cloned neuronal transporter (VMAT2) by expression of the cloned cDNAs in heterologous systems, then determine the relationship to dopamine release. Specific Aim 1: Establish a role for transporter reversal in vesicular efflux, exchange and amphetamine action. Specific Aim 2: Examine the regulation of VMAT2. Specific Aim 3: Determine the effect of activity on intracellular trafficking of VMAT2. We will then extend these studies to primary neuronal cultures and in collaboration with Dr. Sulzer, use voltammetry to determine the role of VMAT2 in regulating quantal size.